Method of synthesising sulforaphane

ABSTRACT

The present invention relates to a method of synthesising sulforaphane by reacting a compound of formula (A) with an oxidizing agent in an aqueous solvent and in the presence of a catalyst. The invention further provides a method of synthesising a stabilised complex of sulforaphane and cyclodextrin by mixing the sulforaphane prepared by the methodology defined herein with cyclodextrin in an aqueous solvent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation application under 35 U.S.C. §120 ofU.S. application Ser. No. 16/429,726, filed Jun. 3, 2019, which is acontinuation application of Ser. No. 15/392,370, filed Dec. 28, 2016,now U.S. Pat. No. 10,307,390, which is a continuation of U.S.application Ser. No. 14/404,773, filed Dec. 1, 2014, now U.S. Pat. No.9,567,405, which is a national stage entry under 35 U. S.C. §371 ofPCT/GB2013/051458, filed May 31, 2013, which claims priority to U.S.Provisional Application No. 61/654,277, filed on Jun. 1, 2012, thedisclosures of which are incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

The present invention relates to a method of synthesising sulforaphane.The present invention also relates to a method of synthesising astabilised sulforaphane-cyclodextrin complexes.

BACKGROUND OF THE INVENTION

According to the US National Cancer Institute, sulforaphane isconsidered to be one of the 40 most promising anticancer agents (KelloffG. J, Crowell J. A, Steele V. E, Lubet R. A, Malone W. A, Boone C. W,Kopelovich L, Hawk E. T, Lieberman R, Lawrence J. A, Ali I, Viner J. L,Sigman C. C, J. Nutr, 2000, 130, 467). It is also known to possessantimicrobial properties. Suforaphane has therefore attracted interestas a potential agent for the treatment and/or prevention of cancer andmicrobial infections.

Sulforaphane is found in the cruciferous vegetables such as cabbage,broccoli, broccoli sprouts, brussel sprouts, cauliflower, cauliflowersprouts, bok choy, kale, collards, arugula, kohlrabi, mustard, turnip,red raddish, and water cress. In the plant, it is present in bound formas glucoraphanin, a glucosinolate. In nature, sulforaphane is oftenformed from glucoraphanin following plant cell damage by an enzymaticreaction.

Various synthetic methods of producing sulforaphane are known in theart. Sulforaphane was synthesized as early as 1948 by Schmid and Karrer(Schmid H. And Karrer, P.; Helvetica Chimica Acta. 1948; 31; 6:1497-1505). The Schmid synthesis results in a racemic mixture.

Various alternative synthetic procedures have been reported by, forexample, Vermeulen and co-workers (Vermeulen M, Zwanenburg B, ChittendenG. J. F, Verhagen H, Eur. J. Med. Chem, 2003, 38(78), 729-737), Conawayand co-workers ((Conaway C. C, Wang C. X, Pittman B, Yang Y. M, SchwartzJ. E, Tian D, McIntee E. J, Hecht S. S, Chung F. L, Cancer Research,2005, 65(18), 8548-8557), Kuhnert and co-workers (Kuhnert N and Lu Y,Journal of Labelled Compounds & Radiopharmaceuticals 2004, 47(8),501-507), Rajski and co-workers (Mays J. R and Rajski, S. R.ChemBioChem, 2008, 9(5), 729-747 and WO2008/008954), Christopher andco-workers (Christopher A. D'Souza, Shantu Amin, Dhimant Desai, Journalof Labelled Compounds & Radiopharmaceuticals, 2003, 46(9), 851-859),Takayuki and co-workers (Joon-Kwan M, Jun-Ran K, Young-Joon A andTakayuki S, Journal of Agricultural and Food Chemistry 2010, 58 (11),6672-6677), and Rabhi and co-workers (WO 2008015315 and US 0135618 A1),Cao and his co-workers (Tong Jian Ding, Ling Zhou, Xiao Ping Cao,Chinese Chemical Letters, 2006, 17(9), 1152-1154) and Chen andco-workers (Xin Chen, Zhengyi Li, Xiaoiang Sun, Hongzhao Ma, XiaoxinChen, Jie Ren, Kun Hu, Synthesis, 2011, 24, 3991-3996 and CN 102249968).

Although sulforaphane has been synthesized by various different methods,most of the reported methods suffer several drawbacks; such as, forexample, low yields, the use of hazardous and potentially harmfulreagents (such as thiophosgene which is a highly toxic and volatileliquid with an unpleasant and irritating odour), the use of Class I orII solvents, laborious work-up/purification procedures and unwantedby-products (such as the inseparable disulfone/sulfonyl derivative ofsulforaphane). These processes are therefore not suitable for theefficient large-scale synthesis of suforaphane.

Therefore, there is a need for an alternative process for synthesisingsulforaphane which address one or more of the aforementioned drawbacksof the prior art processes. In particular. There is a need for asynthetic process that can be implemented on a large scale. Such aprocess will ideally be:

(i) efficient, i.e. it gives commercially viable yields of sulforaphanewith good levels of purity and utilises a small number of syntheticsteps;

(ii) cost effective, i.e. utilising low cost reactants and reactionconditions;

(iii) environmentally acceptable and safe to implement, i.e. it does notuse overly toxic reagents or solvents.

One further and significant problem associated with sulforaphane is itsinherent instability. Sulforaphane exists in the form of an unstable oilwhich rapidly degrades under normal conditions. This makes sulforaphaneexceptionally hard to manufacture and distribute.

Therefore, there is also a need for a process of synthesisingsulforaphane that is readily amenable to subsequent processing steps toprovide a more stable form of the sulforaphane that is produced.

One particularly effective approach to stabilise sulforaphane involvesthe formation of sulforaphane-cyclodextrin complexes. In this regard,U.S. Pat. No. 7,879,822B2, the entire contents of which are herebyincorporated by reference, describes the preparation ofsuforaphane-cyclodextrin complexes having good stability.

It therefore a further object of the present invention to provide afacile process that enables the sulforaphane that it synthesized to bereadily stabilized by the formation of a sulforaphane-cyclodextrincomplex.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention, there is provideda method of synthesising sulforaphane, the method comprising:

reacting, in an aqueous solvent, a compound of formula A:

with an oxidizing agent in the presence of a catalyst.

The process of the present invention possesses a number of advantagesover conventional prior art methods of producing sulforaphane. Firstly,the process is efficient and provides high yields of the desiredsulforaphane end product without significant production of the sulfonylderivative (Erysolin) as a by-product. Secondly, the reaction canproceed under mild, aqueous conditions. The use of an aqueous solvent,such as water, is particularly advantageous because it avoids the use ofexpensive and/or potentially hazardous solvents. It also enables the useof less hazardous oxidising agents, such hydrogen peroxide. In addition,the use of an aqueous solvent makes the reaction ideally suited for thequick and efficient in situ formation of stabilisedsulforaphane-cyclodextrin complexes (by simply mixing the sulforaphaneend product with an aqueous solution of cyclodextrin to form thecomplex).

The process of the present invention is also suited to large-scalemanufacture of suforaphane.

In another aspect, the present invention relates to sulforaphane formedby, obtainable by, obtained by, or directly obtained by, a process asdefined herein.

In yet another aspect, the present invention there is provided a processfor the preparation of a complex of sulforaphane and cyclodextrin, theprocess comprising:

(i) reacting, in an aqueous solvent, a compound of formula A:

with an oxidizing agent in the presence of a catalyst to formsulforaphane; and mixing the sulforaphane from step (i) withcyclodextrin in an aqueous solvent to form a precipitate of thesulforaphane-cyclodextrin complex.

In yet another aspect, the present invention provides asulforaphane-cyclodextrin complex formed by, obtainable by, obtained by,or directly obtained by, a process as defined herein.

In another aspect, the present invention relates to asulforaphane-cyclodextrin complex as defined herein for use in thetreatment and/or prevention of microbial infections and/or cancer.

In another aspect, the present invention provides a method of treatingand/or preventing microbial infections and/or cancer, the methodcomprising administering to an individual in need of such treatment atherapeutically effective amount of a sulforaphane-cyclodextrin complexas defined herein.

In another aspect, the present invention relates to a pharmaceuticalcomposition comprising a sulforaphane-cyclodextrin complex as definedherein and one or more additional pharmaceutical excipients.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless otherwise stated, the following terms used in the specificationand claims have the following meanings set out below.

A “therapeutically effective amount” means the amount of the compoundthat, when administered to a subject for treating a disease or conditionreferred to herein, is sufficient to effect such treatment for thedisease or condition. The “therapeutically effective amount” will varydepending on the form of the compound (e.g. the salt form), the diseaseor condition concerned and its severity, as well as the age, weight,etc., of the subject to be treated.

The term “individual” is used herein to mean a warm blooded mammal.Thus, the compound of the present invention may be used for human and/orveterinary applications. In a particular embodiment, the subject is ahuman.

The term Erysolin is used herein to refer to a compound having thestructure shown below:

ErysolinThe Process of Producing Sulforaphane

It will be appreciated that, in the description of the synthetic methodsdescribed herein, all proposed reaction conditions, including the choiceof the aqueous solvent, the reaction atmosphere, the reactiontemperature, the duration of the experiment and any workup proceduresemployed, can be selected by a person skilled in the art. It will alsounderstood by one skilled in the art of organic synthesis that thefunctionality present on various portions of the molecule must becompatible with the reagents and reaction conditions utilised.

As indicated above, the present invention provides a method ofsynthesising

sulforaphane, the method comprising:

reacting, in an aqueous solvent, a compound of formula A:

with an oxidizing agent in the presence of a catalyst.

The resultant sulforaphane compound has the structure shown below:

The sulforaphane can be collected and appropriately stored forsubsequent use or, more preferably, it can be mixed directly or in situwith cyclodextrin to form a stabilised sulforaphane-cyclodextrin complexas defined further herein. This avoids the need for laboriouspurifications of the sulforaphane end product.

Any suitable aqueous solvent may be used for the reaction. In anembodiment of the invention, the solvent is water, but mixtures of waterand one or more water miscible solvents may also be used in certaincircumstances.

Suitably, the aqueous solvent is degassed prior to the reaction. Anysuitable procedure known in the art for degassing the aqueous solventmay be used. For example, the solvent may be degassed by sparging thesolvent with an inert gas (such as nitrogen or argon), refluxing thesolvent, or by utilising vacuum or ultrasonic degassing procedures.

Any suitable oxidising agent may used in the reaction, provided that itis capable of oxidising the compound of formula A to sulforaphane in anaqueous environment. For example, the oxidising agent may be selectedfrom hydrogen peroxide or water soluble or miscible organic per-acids,such as meta-Chloroperoxybenzoic acid (mCPBA). In an embodiment, theoxidising agent is hydrogen peroxide. Hydrogen peroxide is particularlysuitable because it reacts in the methodology of the present inventionto form sulforaphane and water as the end products (i.e. no unwantedby-products are formed).

Suitably, the oxidising agent is present in an amount sufficient tooxidise all of the compound of formula A to sulforaphane. Typically,about one molar equivalent (relative to the compound of formula A) ofthe oxidising agent will be required, although it is possible to use aslight excess of the oxidizing agent if the conditions are controlled toprevent or limit the formation of the sulfonyl by-product. For example,in some cases, 1 to 2 molar equivalents of oxidizing agent (relative tothe compound of formula A) may be used, more suitably 1 to 1.5 molarequivalents of oxidizing agent is used, and even more suitably 1 to 1.1molar equivalents of oxidizing agent is used.

The reaction also proceeds in the presence of a suitable catalyst. Anycatalyst that is compatible with the aqueous solvent and which iscapable of promoting the oxidation of the compound of formula A may beused. The catalyst must be active in the aqueous environment and may behomogeneous or heterogeneous. Examples of suitable catalysts includeacid catalysts such as cyclodextrin and/or Fuller's Earth, and organicor inorganic acids, such as, for example, ascorbic acid, formic acid,acetic acid, and/or sulphuric acid.

In a particular embodiment the catalyst is cyclodextrin. Any suitablecyclodextrin may be used as the catalyst. For example, the cyclodextrinmay be selected from one or more of W6 (alpha) cyclodextrin (a six sugarring molecule), W7 (beta) cyclodextrin (a seven sugar ring molecule), W8(gamma) cyclodextrin (an eight sugar ring molecule), derivatives thereof(such as hydroxyalkyl derivatives, e.g. hydroxypropyl cyclodextrin), andmixtures thereof. Other cyclodextrins known in the art are alsocontemplated as useful in the synthetic method and the invention shallnot be limited to the specific cyclodextrins listed.

In an embodiment of the invention, the cyclodextrin used as a catalystis alpha-cyclodextrin.

The amount of catalyst required will vary depending on the nature of theoxidizing agent, catalyst and the reaction conditions used. Suitably,0.0001 to 1.0 molar equivalents of catalyst are present (relative to thecompound of formula A) and, more suitably, 0.005 to 0.2 molarequivalents of catalyst are present, and even more suitably, 0.005 to0.05 molar equivalents of catalyst are present.

In embodiments of the invention where the oxidizing agent is hydrogenperoxide, the hydrogen peroxide is suitably added to the reactionmixture slowly and the temperature of the reaction mixture is maintainedat 15° C. or less or, more preferably, 10° C. or less. Suitably, thetemperature is monitored during the addition of the hydrogen peroxideand the rate of addition is adjusted to ensure the temperature remainswithin the desired limits.

In an embodiment of the invention, the oxidizing agent is hydrogenperoxide, the solvent is water, and the catalyst is selected fromcyclodextrin, Fuller's Earth, and acids such as ascorbic acid, formicacid, acetic acid, and/or sulphuric acid. In a particular embodiment ofthe invention, the oxidizing agent is hydrogen peroxide, the solvent iswater, and the catalyst is cyclodextrin, particularly α-cyclodextrin. Insuch embodiments, the compound of formula A and the catalyst may bedissolved in the water and cooled to less than 15° C. or, morepreferably, less than 10° C. (for example, between 1 and 2° C.) and theaqueous hydrogen peroxide solution may then added to the cooled solutionin a controlled manner so that temperature does not exceed 15° C., or,more preferably, 10° C. The reaction may then be stirred and allowed toproceed for a suitable time, for example between 1 and 48 hours. Thesuforaphane product may then be collected or used in subsequent processsteps.

The starting material, i.e. the compound of formula A, can be sourcedcommercially (it can be obtained from various suppliers as either anatural or synthetic product) and/or prepared by techniques known in theart. For example, the compound of formula A may be prepared as anintermediate from a compound of formula B shown below

by procedures described by Vermeulen and co-workers (Eur. J. Med. Chem,2003, 38(78), 729-737), D'Souza and co-workers (Journal of LabelledCompounds & Radiopharmaceuticals, 2003, 46(9), 851-859), Cao and hisco-workers (Chinese Chemical Letters, 2006, 17(9), 1152-1154) and Chenand co-workers (Synthesis, 2011, 24, 3991-3996 and CN 102249968).

In an embodiment of the present invention, the compound of formula A isprepared by reacting a compound of the formula B

with carbon disulphide in a suitable solvent (e.g. THF) and in thepresence of a suitable base (such as Et₃N) and a suitable oxidizingagent (such as hydrogen peroxide).

In a particular embodiment, the solvent is THF, the base istriethylamine, and the oxidizing agent is hydrogen peroxide.

In a further embodiment, the compound of formula B and the base (e.g.triethylamine) are dissolved in a solvent (e.g. THF) at a lowtemperature (e.g. below 25° C., more preferably below 0° C. and, evenmore preferably, below −10° C.). Carbon disulphide is then added to thereaction mixture. Suitably the temperature is controlled while thecarbon disulphide is added (e.g. it is kept below 25° C., or morepreferably below 5° C. and, even more preferably, below 0° C.). Carbondisulphide may be added at a controlled rate in order to keep thetemperature of the reaction mixture low (e.g. it may be added drop wiseover a period of, for example, 0.5 to 4 hours). The reaction mixture maythen be warmed (for example, to between 5 and 25° C., and morepreferably to between 5 and 20° C.) and then the oxidizing agent (e.g.hydrogen peroxide) is added.

The resultant crude product of the compound of formula A can becollected, washed and purified (for example by distillation) to give apure compound of formula A using techniques well known in the art.

The Process of Producing Sulforaphane-Cyclodextrin Complexes

The present invention further provides a process for the preparation ofa complex of sulforaphane and cyclodextrin, the process comprising:

(i) reacting, in an aqueous solvent, a compound of formula A:

with an oxidizing agent in the presence of a catalyst to formsuforaphane; and(ii) mixing the aqueous solution of sulforaphane from step (i) with anaqueous solution of cyclodextrin in an aqueous solvent to form aprecipitate of the sulforaphane-cyclodextrin complex.

Step (i) of the reaction is the process of synthesising sulforaphanedefined above. One particular advantage of using an aqueous solvent instep (i) is that once the reaction is complete, it enables the simpleaddition of an aqueous solution of cyclodextrin to the reaction mixturein order to form a stabilised sulforaphane-cyclodextrin complex.Therefore, the present process provides a simple, effective and rapidmeans by which the sulforaphane can be stabilised.

Sulforaphane-cyclodextrin complexes are described in U.S. Pat. No.7,879,822B2, the entire contents of which are hereby incorporated byreference.

The process of the present invention suitably comprises an additionalstep of collecting the precipitate of the sulforaphane-cyclodextrincomplex and then optionally washing and drying the precipitate. Theprecipitate may be collected by techniques well known in the art, suchas by filtration.

Suitable reaction conditions for forming a sulforaphane-cyclodextrincomplex in aqueous solutions are known in the art from U.S. Pat. No.7,879,822 B2.

In some embodiments, the purity of the resulting complex can be furtherincreased by recrystallization.

Any suitable cyclodextrin may be for forming a complex with thesulforaphane. In embodiments where the catalyst in step (i) iscyclodextrin, then the cyclodextrin used for forming a complex in step(ii) may be the same or different to the cyclodextrin used as a catalystin step (i). By way of example, the cyclodextrin for use in the methodsof the present invention may be selected from one or more of W6 (alpha)cyclodextrin (a six sugar ring molecule), W7 (beta) cyclodextrin (aseven sugar ring molecule), W8 (gamma) cyclodextrin (an eight sugar ringmolecule), derivatives thereof (such as hydroxyalkyl derivatives, e.g.hydroxypropyl cyclodextrin), and mixtures thereof. Other cyclodextrinsknown in the art are also contemplated as useful in the presentprocesses and the invention shall not be limited to the specificcyclodextrins listed.

In an embodiment of the invention, the cyclodextrin used for forming acomplex with the sulforaphane in step (ii) is alpha-cyclodextrin.

Prior to mixing with the sulforaphane obtained from step (i), thecyclodextrin utilized in step (ii) of the present method may bedissolved in an aqueous solvent, such as water. Thedissolution/dispersion of cyclodextrin in the solvent may beaccomplished by any method known in the art. For example, in someembodiments, the cyclodextrin may be fully or partially dissolved in anaqueous solvent by placing the cyclodextrin in the solvent and heatingthe mixture. In additional embodiments, sonication may be utilized toeither fully or partially dissolve the cyclodextrin in the solvent. Infurther embodiments, multiple methods of dissolution may be utilized toreach the level of dissolution desired by the user, for example, byutilizing sonication in connection with heating the solvent.

Once the sulforaphane and cyclodextrin have been added together in step(ii) of the process, and are ready to be mixed, any method of mixing maybe utilized. For example, the components may be mixed by stirring,sonication, agitation, or other methods known in the art. In someembodiments, more than one method of mixing may be utilized together.

The duration of the mixing may vary based on the particular methods ofmixing utilized. For example, if stirring or sonication is utilized, thesulforaphane, and cyclodextrin may be mixed for from about 2 hours toabout 48 hours. In other embodiments, the sulforaphane and cyclodextrinmay be mixed by a stirrer or sonication for about 6 hours to about 15hours.

As discussed above, multiple methods of mixing may be utilized formixing the sulforaphane and cyclodextrin. For example, in someembodiments, sonication may be utilized in connection with stirring. Insuch embodiments, sonication may be utilized for a time period of fromabout 0.01 hours to about 1.5 hours during mixing with a stirrer forfrom about 2 hours to about 48 hours.

The initial mixing of the suforaphane and the cyclodextrin at ambienttemperature, for example between 15° C. and 25° C. However, in aparticular embodiment, after the sulforaphane and cyclodextrin have beenmixed, the mixture is cooled to stabilize the formed precipitate. Theparticular sulforaphane and cyclodextrin used may dictate the durationand severity of the cooling required. For example, the mixture may becooled to a temperature within the range of about −10° C. to about 20°C., more suitably between about −8° C. to about 10° C., even moresuitably between about −5° C. to about 4° C. The duration of the coolingcan vary and may be, for example, from about 0.1 hours to about 24hours.

In a particular embodiment, the mixture may be cooled to a temperaturefrom about −5° C. to about 2° C., optionally for a time period of about0.5 hour to about 4 hours. The precipitate may then be filtered toobtain a sulforaphane-cyclodextrin complex of increased purity.

Suitably the molar ratio of sulforaphane to cyclodextrin in theresultant complex is within the range of 0.4:1 to 1:1; suitably 0.8:1 to1:1; and more suitably 0.9:1 to 1:1, 0.95:1 to 1:1 or 0.98:1 to 1:1.

In further embodiments, the resulting complex may be recrystallized toobtain a complex with an even greater purity level of the sulforaphane.In such embodiments, any method of recrystallization known in the artmay be utilized. For example, in some embodiments, recrystallization maybe accomplished by cooling the resulting mixture, by dissolving theresulting mixture in a second solvent, through a chemical reaction, bychanging the pH of the mixture or by evaporating the solvent. The user'sspecifications may dictate the particular methods utilized.

In some embodiments, the method of recrystallization may includedissolution of the formed solid particles in a solvent. Such dissolutionmay be completed by any method known in the art. For example, in someembodiments, the dissolution may be completed through sonication. Thesonication may be completed at an elevated temperature, i.e. from about50° C. to about 100° C., and may be continued until no solid particlesremain. Additionally, any solvent known in the art may be utilized,including those indicated above that may be useful in connection withdissolving cyclodextrin.

After dissolution has been substantially completed, the mixture may beheld at room temperature to allow the solids to precipitate out ofsolution. Depending on the materials utilized, the time in which themixture is held at room temperature may vary. For example, ifsulforaphane is utilized, most of the solids may precipitate out ofsolution within an hour of being held at room temperature. In otherembodiments, the solution may take more than or less than an hour tosufficiently allow the solids of the complex to precipitate out ofsolution.

As discussed above, the solids may then be cooled to aid the formationand stabilization of the complex. The particular complex used maydictate the amount of cooling necessary. For example, in someembodiments, the mixture may be cooled in a cooling device, such as forexample a refrigerator, that is maintained at a temperature from about−10° C. to about 20° C., optionally for a time from about 0.1 hours toabout 2 hours. In other embodiments, the mixture may be cooled in acooling device that is maintained at a temperature from about 2° C. toabout 6° C. for a time between about 0.5 hours to 1 hour. After thecomplex has sufficiently crystallized, it may then be filtered toproduce a sulforaphane-cyclodextrin complex of even greater purity.

Pharmaceutical Compositions and Methods of Treatment

In another aspect, the invention is directed to a method of providinganticancer and/or antimicrobial treatments to a subject in need of suchtreatment. The method includes administering to a subject in need ofsuch treatment the sulforaphane-cyclodextrin complexes of increasedpurity defined herein in a therapeutically effective amount.

A first component of the treatment method is sulforaphane-cyclodextrincomplex prepared in accordance with the methods defined herein. Thecomponents that are useful in the present invention can be of any purityor grade, as long as the preparation is of a quality and stabilitysuitable for pharmaceutical use and does not affect the resultingpreparation's physiological activity or safety.

The method may further include administration of other pharmaceuticallyacceptable components. The term “pharmaceutically acceptable” is usedadjectivally herein to mean that the modified noun is appropriate foruse in a pharmaceutical product.

When the sulforaphane-cyclodextrin complex created by the presentmethods is supplied along with a pharmaceutically acceptable carrier orpharmaceutically acceptable excipient, which terms can be usedinterchangeably herein, a pharmaceutical composition may be formed. Thepharmaceutical compositions of the invention may be prepared by any ofthe well-known techniques of pharmacy, for example, by admixing thecomponents.

A pharmaceutical composition of the present invention is directed to acomposition suitable for the prevention or treatment of the disordersdescribed herein.

Pharmaceutically acceptable carriers and excipients are chosen such thatside effects from the pharmaceutical compound(s) are minimized and theperformance of the compound(s) is not canceled or inhibited to such anextent that treatment is ineffective. Pharmaceutically acceptablecarriers include, but are not limited to, physiological saline,Ringer's, phosphate solution or buffer, buffered saline, and othercarriers known in the art. Pharmaceutical compositions may also includestabilizers, anti-oxidants, colorants, and diluents.

The carrier should be acceptable in the sense of being compatible withthe other ingredients of the composition and not be deleterious to therecipient. The carrier can be a solid or a liquid, or both, and may beformulated with the compound(s) as a unit-dose composition, for example,a tablet, which can contain from about 0.01% to about 95% by weight ofthe active compound(s).

The pharmaceutically acceptable carrier can also be selected on thebasis of the desired route of administration of the compound(s). Thedesired route of administration may be one or more of oral, enteral,parenteral, injectable, buccal, and topical. For example, in anembodiment, the carrier is suitable for oral administration. In someembodiments, the composition includes a carrier or additional agent thatis suitable for promoting delivery of the compound(s) to thegastrointestinal or intestinal tract.

In particular, the pharmaceutical compositions of the present invention,or compositions in which they are included, can be administered orally,for example, as tablets, coated tablets, dragees, troches, lozenges,aqueous or oily suspensions, dispersible powders or granules, emulsions,hard or soft capsules, or syrups or elixirs. Compositions intended fororal use may be prepared according to any method known in the art forthe manufacture of pharmaceutical compositions and such compositions maycontain one or more agents selected from the group consisting ofsweetening agents, flavoring agents, coloring agents and preservingagents in order to provide pharmaceutically acceptable and palatablepreparations. Tablets may contain the active ingredient in admixturewith non-toxic pharmaceutically acceptable excipients which are suitablefor the manufacture of tablets. These excipients may be, for example,inert diluents, such as calcium carbonate, sodium carbonate, lactose,calcium phosphate or sodium phosphate; granulating and disintegratingagents, for example, maize starch or alginic acid; binding agents, forexample starch, gelatin, or acacia, and lubricating agents, for examplemagnesium stearate, stearic acid or talc. The tablets may be uncoated orthey may be coated by known techniques to delay disintegration andadsorption in the gastrointestinal tract and thereby provide a sustainedaction over a longer period. For example, a time delay material such asglyceryl monostearate or glyceryl distearate may be employed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredients are mixed with an inert solid diluent,such as for example calcium carbonate, calcium phosphate or kaolin, oras soft gelatin capsules wherein the active ingredients are present ormixed with water or an oil medium, such as for example peanut oil,liquid paraffin, any of a variety of herbal extracts, milk, or oliveoil.

Aqueous suspensions can be produced that contain the active materials inadmixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients include suspending agents, such as forexample sodium carboxymethylcellulose, methylcellulose,hydroxypropylmethyl-cellulose, sodium alginate, polyvinylpyrrolidone gumtragacanth and gum acacia; dispersing or wetting agents may benaturally-occurring phosphatides, such as for example lecithin, orcondensation products of an alkylene oxide with fatty acids, such as forexample polyoxyethylene stearate, or condensation products of ethyleneoxide with long chain aliphatic alcohols, such as for exampleheptadecaethyleneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol, such as forexample polyoxyethylene sorbitol monooleate or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides, such as for example polyoxyethylene sorbitan monooleate.

The aqueous suspensions may also contain one or more preservatives, suchas for example ethyl or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, or one or more sweetening agents,such as sucrose, glycerol, sorbitol or saccharin.

Oily suspensions may be formulated by suspending the active ingredientsin an omega-3 fatty acid, a vegetable oil, such as for example arachisoil, olive oil, sesame oil or coconut oil, or in a mineral oil such asliquid paraffin. The oily suspensions may contain a thickening agent,such as for example beeswax, hard paraffin or cetyl alcohol.

Sweetening agents, such as those set forth above, and flavoring agentsmay be added to provide a palatable oral preparation. These compositionsmay be preserved by the addition of an antioxidant such as ascorbicacid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, a suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present.

Syrups and elixirs containing the sulforaphane-cyclodextrin complex maybe formulated with sweetening agents, such as for example glycerol,sorbitol, or sucrose. Such formulations may also contain a demulcent, apreservative, and/or flavoring and coloring agents. Liquid dosage formsfor oral administration can include pharmaceutically acceptableemulsions, solutions, suspensions, syrups, and/or elixirs containinginert diluents commonly used in the art, such as water. Suchcompositions may also comprise adjuvants, such as wetting agents,emulsifying and/or suspending agents, and sweetening, flavoring, and/orperfuming agents.

Pharmaceutical compositions suitable for oral administration can bepresented in discrete units each containing a predetermined amount of atleast one therapeutic compound useful in the present invention; as apowder or granules; as a solution or a suspension in an aqueous ornonaqueous liquid; or as an oil-in-water or water-in-oil emulsion. Asindicated, such compositions can be prepared by any suitable method ofpharmacy, which may include the step of bringing into association theactive compound(s) and the carrier (which can constitute one or moreaccessory ingredients). In general, the compositions are prepared byadmixing the active compound with a liquid or finely divided solidcarrier, or both, and then, if necessary, shaping the product.

For example, a tablet can be prepared by compressing or molding a powderor granules of the compound, optionally with one or more accessoryingredients. Compressed tablets can be prepared by compressing, in asuitable machine, the compound in a free-flowing form, such as a powderor granules optionally mixed with a binder, lubricant, inert diluentand/or surface active/dispersing agent(s). Molded tablets can be made bymolding, in a suitable machine, the powdered compound moistened with aninert liquid diluent.

Oral delivery of the combinations of the present invention can includeformulations, as are well known in the art, to provide prolonged orsustained delivery of the drug to the gastrointestinal and/or intestinaltract by any number of mechanisms. These include, but are not limitedto, pH-sensitive release from the dosage form based on the changing pHof the small intestine, slow erosion of a tablet or capsule, retentionin the stomach based on the physical properties of the formulation,bioadhesion of the dosage form to the mucosal lining of the intestinaltract, or enzymatic release of the active drug from the dosage form. Forsome of the therapeutic compounds useful in the methods, combinationsand compositions of the present invention, the intended effect is toextend the time period over which the active drug molecule is deliveredto the site of action by manipulation of the dosage form. Thus,enteric-coated and enteric-coated controlled release formulations arewithin the scope of the present invention. Suitable enteric coatingsinclude cellulose acetate phthalate, polyvinylacetate phthalate,hydroxypropylmethylcellulose phthalate and anionic polymers ofmethacrylic acid and methacrylic acid methyl ester.

In certain embodiments, the pharmaceutical composition may includetablets that may be uncoated or they may be coated by known techniquesto delay disintegration and absorption in the gastrointestinal tract andthereby provide a delayed action over a longer period. For example, atime delay material such as glyceryl monostearate or glyceryl distearatemay be employed.

In additional embodiments, the compositions created by the subjectmethod may be administered parenterally, such as for examplesubcutaneously, intravenously, intramuscularly, intrasternally, or byinfusion techniques, in the form of sterile injectable aqueous orolagenous suspensions. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxic parenterallyacceptable diluent or solvent, such as for example a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed, including synthetic mono- or diglycerides. In addition,n-3 polyunsaturated fatty acids may find use in the preparation ofinjectables.

Pharmaceutical compositions suitable for parenteral administration cancomprise sterile aqueous preparations of a compound of the presentinvention. These preparations may be administered intravenously,although administration can also be effected by means of subcutaneous,intramuscular, or intradermal injection or by infusion. Suchpreparations may be prepared by admixing the compound with water andrendering the resulting solution sterile and isotonic with the blood.Injectable compositions according to the invention will generallycontain from 0.01 to 10% w/w of a compound disclosed herein.

The active ingredients may also be administered by injection as acomposition wherein, for example, saline, dextrose, or water may be usedas a suitable carrier. A suitable daily dose of each active therapeuticcompound is one that achieves relatively the same blood serum level asproduced by oral administration as described above.

Also encompassed by the present invention is buccal or “sub-lingual”administration, which includes lozenges or a chewable gum comprising thecompounds set forth herein. The compounds can be deposited in a flavoredbase and acacia or tragacanth or the compounds may be deposited inpastilles comprising the compounds in an inert base such as gelatin andglycerin or sucrose and acacia.

The pharmaceutical compositions of the present invention are alsosuitable for topical application to the skin and may take the form ofointments, creams, lotions, pastes, gels, sprays, powders, jellies,collyriums, solutions, suspensions, aerosols, or oils. Carriers may beused and include petroleum jelly (e.g., Vaseline), lanolin, polyethyleneglycols, alcohols, and combinations of two or more thereof. The activecompound or compounds are generally present at a concentration of from0.01 to 50% w/w of the composition, such as for example from about 0.01to about 2%.

The present invention may also include safe and effective amounts ofisotonicity agents, including, salts, such as sodium chloride, and/ornon-electrolyte isotonicity agents such as sorbitol and mannitol.

The solubility of the components of the present compositions may beenhanced by a surfactant or other appropriate co-solvent in thecomposition. Such co-solvents include polysorbate 20, 60, and 80,polyoxyethylene/polyoxypropylene surfactants (e.g., Pluronic F-68, F-84and P-103, available from BASF®), cyclodextrin, or other agents known tothose skilled in the art. Such co-solvents may be employed at levels offrom about 0.01% to about 2% by weight.

Pharmaceutically acceptable excipients and carriers encompass all theforegoing and the like. Effective formulations and administrationprocedures are well known in the art and are described in standardtextbooks. See e.g. Gennaro, A. R., Remington: The Science and Practiceof Pharmacy, 20^(th) Edition, (Lippincott, Williams and Wilkins), 2000;Hoover, John E., Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa., 1975; Liberman, et al., Eds., Pharmaceutical DosageForms, Marcel Decker, New York, N. Y., 1980; and Kibbe, et al., Eds.,Handbook of Pharmaceutical Excipients (3 Ed.), American PharmaceuticalAssociation, Washington, 1999.

In the present method, a subject in need of treatment and/or preventionof the disorders described herein and/or related conditions may betreated with an amount of the presently inventive purified sulforaphane,wherein the amount of the individual components provides a dosage oramount that is sufficient to constitute a treatment or preventioneffective amount.

The effective amount of purified sulforaphane-cyclodextrin complex, ofcourse, depend on a number of factors, such as the specific compoundchosen, the use for which it is intended, the mode of administration,the host to be treated, and the clinical condition of the recipient.

A carcinogenic, tumorigenic, or anti-bacterial symptom is consideredameliorated or improved if any benefit is achieved, no matter howslight.

Dosages for the present compositions and methods provided herein may bedetermined and adjusted based on the efficacy demonstrated in providinga chemoprotective or chemopreventative result. In addition, one ofordinary skill in the art will know how to measure and quantify thepresence or absence of carcinogenesis or tumorigenesis symptoms.

Dosages for the present compositions are those that are effective toprovide a chemoprotective, chemopreventative, and/or anti-bacterialeffect.

Those skilled in the art will appreciate that dosages may also bedetermined with guidance from Goodman & Gilman's The PharmacologicalBasis of Therapeutics, Ninth Edition (1996), Appendix II, pp. 1707-1711.

EXAMPLES

The invention will now be illustrated in the following Examples.

General Materials and Methods

¹H and ¹³C NMR spectra were recorded on a Oxford 400 MHz spectrometerusing TMS as the internal standard and the chemical shifts are reportedin ppm.

Electrospray ionization mass spectrometry (ESI-MS) was performed on aMicromass Platform LCZ connected to Waters 2695 separations module andWater 996 photodiode array detector. GC-MS spectrometry was performed ona Agilent 7820A/5975 MSD series.

HPLC was performed on a HP 1050 Module, Column: Phenomenex Gemini C18,5μ, 110A°, 250×4.6 mm. Total run time: 40 min. MeCN in H₂0+0.1% TFA.Flow: 1.5 mL/min.

Detector 244 nm (VWD).

Karl Fischer (H₂0 content) analysis was performed on a KF coulometer 831equipped with Ti stand 703.

All reactions were run under an atmosphere of dry nitrogen and thereported yields are isolated yields. All chemical reagents werepurchased from commercial sources and used as received.

Preparation of Startinq Materials

Preparation of 1-isothiocyanato-4-methylthiobutane (Formula A)

A 50-L multi-neck round bottom flask equipped with an overhead stirrer,a temperature probe and a 1 L addition funnel and a positive flow of N₂was cooled to −10° C. in MeOH/ice bath and charged with THF (EMD,reagent grade, 15.0 L). 1-Amino-4-methylthiobutane (Formula B; 1.5 Kg,12.6 moles, 1.0 equiv.) and triethylamine (1.75 L, 1.0 equiv.) wereadded, and the solution was further stirred until it had cooled below−10° C. Carbon disulfide (755 mL, 1.0 equiv.) was added dropwise over 2hours while keeping the internal temperature below −3° C. (bathtemperature was −20° C.), after which the yellow-green solution had beenwarmed to 11° C. Hydrogen peroxide (35% aq, 1224 mL, 1.0 equiv.) wasadded slowly over 2.5 hours while keeping the internal temperaturebetween 11 to 18° C. (bath temperature was 0° C.), which produced a darkorange-red suspension with swirling yellow particulates.

Workup: After stirring overnight, an aliquot was checked by GC (75°C.→200 at 15°/min, then 40°/min to 300, 2 min hold: 7.73 mins) and thenthe mixture was transferred into a 50-L workup station using a hoseequipped with a filter head. The mixture was diluted with 4.5 L of ethylacetate, and then washed with 10% HCl (6 L), water (6 L), and brine (7.5L). The collected organic layer was dried over anhydrous Na₂SO₄,filtered and concentrated under vacuum to yield ˜2 kg of dark red oil.

Distillation: The red oil was transferred into a 2 L (3 batches) roundbottom flask and connected to the Kugelrohr. The apparatus was placedunder high vacuum (˜0.3-0.5 torr), and the air bath heated to 85° C. Theforerun (mostly ethyl acetate and trace unknown by-product) wasdiscarded. After changing the receiver, the bath temperature wasincreased to 115° C. Pale yellow material distilled over at 100-110° C.,and immediately froze upon contact with the dry-ice/acetone bath. Afterdistillations (three batches) yielded 1.7 Kg (84% yield) material at 98%pure by HPLC and >99% pure by GC.

¹HNMR (CDCl₃, 400 MHz); 1.7-1.85 (m, 4H), 2.2 (s, 3H), 2.55 (t, 2H),3.56 (t, 2H)

Example 1—Preparation of sulforaphane(I-isothiocvanato-4-methylsulfinylbutane)

A 5-L multi-neck round bottom flask equipped with an overhead stirrer, atemperature probe and a 500 mL addition funnel was set-up with apositive flow of N₂. α-Cyclodextrin (30 g, 0.03 moles, 0.01 equivalents)was dissolved in 1 L of distilled water and degassed over 30 minutes bypurging with nitrogen. To the above solution was added 50 g (3.1 moles,1 equivalent) of 1-isothiocyanato-4-methylthiobutane (Formula A) anddegassed again at 0° C. over 30 minutes. To this biphasic reactionmixture was added 305 mL of H₂0₂ (3.1 moles, 1 equivalent, 35% aq.)slowly while maintaining temperature between 0-2° C. [NOTE: Peroxide wasdropped in at a rate sufficiently low so that the temperature did notincrease above 10° C.]. Once the addition complete, the reaction mixturewas stirred at ice bath temperature for about 8 hours and then slowlyallowed to come to room temperature overnight. Reaction mixture wasfiltered to remove the light yellow insoluble solids and then thefiltrate was kept in the refrigerator for ˜1 h. Based on the analyticalHPLC the crude sulforaphane was ˜95% pure.

This material was used for the complexation step (Example 2) without anyfurther workup/purification.

Summary of Three Repetitions—

Lot Reaction size Purity by HPLC (crude) Observations 1 501 g  95% H₂O₂was added at <2° C. 2 500 g 95.6% H₂O₂ was added at <4° C. 3 500 g 95.4%H₂O₂ was added at <2° C.

All three batches were conducted at the same reaction scale and thereactions proceeded in similar way in terms of reaction time and productpurity.

¹HNMR (CDCl₃, 400 MHz); δ1.90 (m, 4H), 2.58 (s, 3H), 2.75 (m, 2H), 3.60(t, 2H).

¹³CNMR (CDCl₃, 100 MHz); δ130.2, 53.4, 44.5, 38.5, 29.5, 20.1

Example 2—Preparation of Sufforaphane-Cyclodextrin Complex

α-Cyclodextrin (Wacker CAVAMAX6 Food Grade, 3015 g, 3.1 moles, 1equivalent) was dissolved in distilled water (8 L) by heating up to 55°C. under nitrogen atmosphere. The homogeneous solution was cooled downto ˜25° C. using an ice-water bath and then degassed for ˜20 min bypurging nitrogen. After degassing, it turned into a foggy solution.Aqueous solution of sulforaphane was removed from the refrigerator (seeprevious step) and then added to the above foggy α-cyclodextrin solutionat once. At this stage reaction temperature was ˜18° C., and continuedstirring at room temperature overnight (˜16 h). The heterogeneousreaction mixture was coaled down to 1-2° C. using ice-methanol bath andstirred for 3 hr at that temperature. The precipitated white solid wasfiltered and dried overnight under high vacuum at room temperature bycovering the filter funnel with a latex sheet. The white filter cake wastransferred into a 10-L rotovap flask and dried further at roomtemperature under a high vacuum to afford 2,802 g of complex (98.7% pureby HPLC, 78.5% yield).

Summary of Three Repetitions—

Lot Reaction size** Purity by HPLC Yield* 1 550.8 g 98.5% 78.5% 2 549.7g 98.6% 76.9% 3 549.7 g 98.7% 73.2% *overall yield in last two steps,**based on the 100% conversion in the previous step.

All three batches were conducted at almost same scale and the reactionsproceeded in similar way in terms of reaction time, yield, productpurity and percentage loading sulforaphane on α-cyclodextrin.

¹HNMR (D₂0, 400 MHz); δ1.99 (br, 4H), 2.73 (s, 3H), 2.98 (br, 2H), 3.60(m, 12H), 3.70 (br, 2H), 3.92 (m, 24H), 5.11 (d, 6H).

¹³CNMR (D₂0, 100 MHz); δ130.05, 101.82, 81.40, 74.05, 71.98, 71.84,60.34, 52.02, 44.94, 37.03, 29.29, 20.08.

Example 3—Preparation of sulforaphane(1-isothiocvanato-4-methylsulfinylbutane) with Different Catalysts andReaction Conditions

General Procedure:

A multi-neck round bottom flask equipped with an overhead stirrer, atemperature probe and an addition funnel was set-up with a positive flowof N₂. Acid catalyst (0.001 to 0.01 equivalents) was dissolved insolvent (water, acetonitrile, acetone etc.) and degassed over 30 minutesby purging with nitrogen. To the above solution was added 1 equivalentof thioether starting material (2) and degassed again at 0 −5° C. over30 minutes. To this biphasic reaction mixture was added 1 equivalent ofoxidizing agent (H₂0₂, m-CBPA etc.) slowly while maintaining temperaturebetween 0-10° C. Once the addition complete, the reaction mixture wasstirred at ice bath temperature for about 8-24 hours and then slowlyallowed to come to room temperature overnight. Reaction mixture wasfiltered to remove the insoluble solids and then the filtrate was keptin the refrigerator or used immediately in the following step. Based onthe analytical HPLC the crude sulforaphane was ≥95% pure. This materialwas used for the complexation step without any further purification.

The oxidation of compound 2 into sulforaphane in various solvents and/orin the presence of different catalysts/acids are shown in the Tablebelow, along with the reaction conditions.

Reaction Conditions H₂O₂/H₂O H₂O₂/H₂O, H₂O₂/H₂O, H₂O₂/H₂O, H₂O₂/H₂O 0.1%H₂O₂/H₂O, H₂O_(2/) H₂O, 0.1eq 0.01eq 1.0 eq 0.05 eq Fuller's Acetone,Acetonitrile α-CD α-CD, α-CD, AcOH Earth 0° C. to RT 0° C. to RT 0° C.to RT 0° C. to RT 0° C. to RT 0° C. to RT 0° C. to RT Purity 95.6% 96.8%96% 98% 77% 98.8% 98% by HPLC (3.1% SM) (0.7% SM) (1% SM) (0.5% SM) (21%S M) (0.06% SM) (0.06 SM) Yield Not Not Not  82%* Not Not Not isolatedisolated isolated isolated isolated isolated Final — — — 98% — — —purity *Isolated yield.

Based on the above results, all of the listed catalysts produced similarresults, but cyclodextrin is preferred since it is used in the nextcomplexation step to stabilize the sulforaphane.

Only trace amount of sulfonyl impurity (Erysolin) was detected by HPLC.

The invention claimed is:
 1. A process for the preparation ofsulforaphane, the process comprising: the formation of a compound offormula A:

from a compound of formula B:

and (ii) reacting, in an aqueous solvent, the compound of formula A withan oxidizing agent, optionally in the presence of a catalyst, to formsulforaphane; wherein in step (ii) the oxidizing agent is hydrogenperoxide.
 2. A process according to claim 1, wherein in step (i) thecompound of formula B is reacted with carbon disulphide in a solvent andin the presence of a base and an oxidizing agent to form the compound offormula A.
 3. A process according to claim 2, wherein the solvent inTHF.
 4. A process according to claim 2, wherein the base istriethylamine.
 5. A process according to claim 2, wherein in step (i)the oxidizing agent is hydrogen peroxide.
 6. A process according toclaim 2, wherein the compound of formula B and the base are dissolved inthe solvent at a temperature below 25° C., before the carbon disulphideis added to the mixture.
 7. A process according to claim 6, wherein thecompound of formula B and the base are dissolved in the solvent at atemperature below 0° C.
 8. A process according to claim 1, wherein instep (i) the compound of formula A is purified.
 9. A process accordingto claim 1, wherein in step (i) the compound of formula A is purified bydistillation.
 10. A process according to claim 9 or claim 9, whereinafter purification the compound of formula A has a purity by HPLC of atleast 98% and/or a purity by GC of greater than 99%.
 11. A processaccording to claim 1, wherein in step (ii) the aqueous solvent is water.12. A process according to claim 1, wherein in step (ii) the catalyst isselected from cyclodextrin and organic or inorganic acids, such asascorbic acid, formic acid, acetic acid, and/or sulphuric acid.
 13. Aprocess according to claim 12, wherein the catalyst is a cyclodextrin.14. A process according to claim 12, wherein the catalyst isalpha-cyclodextrin.
 15. A process according to claim 1, wherein 0.0001to 1.0 molar equivalents of catalyst are present relative to thecompound of formula A.
 16. A process according to claim 1, wherein instep (ii) the temperature of the reaction is maintained at a temperatureof 25° C. or less when the oxidizing agent is added to the reactionmixture.
 17. A process according to claim 16, wherein in step (ii) thetemperature of the reaction is maintained at a temperature of 15° C. orless when the oxidizing agent is added to the reaction mixture.
 18. Aprocess according to claim 1, wherein the sulforaphane has a purity byHPLC of at least 95%.